Methods and compositions for the control of the flesh fly

ABSTRACT

The subject invention concerns novel peptides which have the property of interfering with the biosynthesis of the enzyme trypsin and the biosynthesis of the hormone ecdysone. This property enables the use of these peptides to, for example, inhibit the formation of progeny in blood-ingesting insects, e.g., Neobellieria, since trypsin is an essential enzyme for food digestion which provides the essential building blocks for egg development in such insects.

BACKGROUND OF THE INVENTION

In the grey flesh fly, Neobellieria bullata, vitellogenesis is cyclic.The repeated gonadotropic cycles suggest that egg development is underhormonal regulation. Previously, it has been shown that egg developmentin flies and mosquitoes is regulated by ecdysone and juvenile hormone(Huybrechts and De Loof, 1977, 1981; Hagedorn et al., 1975; Borovsky etal. 1985), which are synthesized by the ovary (Goltzene et al. 1978;Hagedorn et al. 1975; Borovsky et al. 1992a, 1992b, 1993d).Neurosecretory cells in the brain usually produce peptide hormones. Twoof these peptides, EDNH and allatostatin, control the synthesis ofecdysone and juvenile hormone (Hanaoka and Hagedorn, 1980; Woodhead etal., 1989). In addition, a 65-amino acid peptide, ovary maturing parsin,which acts as a true gonadotropin and stimulates vitellogeninbiosynthesis has been recently isolated from the brain of Locustamigratoria (Girardie et al. 1991).

Less information is available about the signals that terminatevitellogenesis. An oostatic factor synthesized by the mosquito ovary wasrecently purified and sequenced from female Aeries aegypti (Borovsky etal. 1990, 1993a). The factor, which is a decapeptide, was named TrypsinModulating Oostatic Factor (TMOF), and its amino acid sequence wasdetermined as NH₂ -YDPAPPPPPP-COOH (See U.S. Pat. No. 5,011,909).However, this peptide was not found in Neobellieria. We found acompletely different hormone in Neobellieria.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns a novel peptide hormone synthesized byand isolated and purified from the ovaries of the grey flesh flyNeobellieria (Sarcophaga) bullata. The subject peptide is useful forinhibiting digestion in pests, thus causing sterility (inhibition of eggdevelopment) in a treated pest. The isolated and purified compounds ofthe subject invention are white powders that are highly soluble inwater. They can be synthesized on a commercial peptide synthesizer.

More specifically, a preferred embodiment of the invention is ahexapeptide from Neobellieria ovaries, which is the trypsin modulatingoostatic factor designated as Neb-TMOF. The subject peptide has thesequence NH₂ -NPTNLH-COOH (SEQ ID NO. 1). Due to the small size of thesubject peptide, it can transverse the gut into the hemolymph muchfaster than the mosquito factor, which has ten amino acids. The site ofaction of these factors is a midgut receptor on the hemolymph side. Thefactor is active at physiological concentrations with an effective doseof 50% inhibition (ED₅₀) of trypsin biosynthesis at 10⁻⁹ M.

Another object of the subject invention is the commercial use of thispeptide to inhibit digestion in flies and other insects. Specifically,the novel factor inhibits trypsin biosynthesis in the midgut bysignalling the midgut cells to stop trypsin biosynthesis after food hasbeen digested. Food digestion in the female fly is essential for thedevelopment of the oocytes (eggs) in the ovary. Without food, or if thefemale fly does not synthesize trypsin after the meal, no eggs will besynthesized in the ovary and female fly is essentially sterile. Femaleflesh flies fed a protein meal and then injected with the novel factorshowed, after 24 hours, 50% to 80% lower trypsin biosynthesis than didcontrol female flesh flies injected with saline.

Following this observation we isolated Neb-TMOF from 10,000 ovaries,purified the hormone, and sequenced it. The purified hexapeptide, atphysiological concentrations of 10⁻⁹ M, inhibited 50% of trypsinbiosynthesis in the midgut of female Neobellieria bullata. Injection ofthe hormone also inhibited egg yolk synthesis because the amino acidsthat are needed for the synthesis were not available when digestionstopped. In addition to its inhibitory effects on trypsin synthesis inthe gut, Neb-TMOF is also a potent inhibitor (EC₅₀ =5×10⁻⁹) of thebiosynthesis of ecdysone by ring glands of the flesh flies Neobellieriaand Calliphora. "EC₅₀ " is recognized in the art to mean the "effectiveconcentration, 50% inhibition," i.e., the concentration at which 50%inhibition is effected. The peptide is also present in larval instars.Accordingly, another object of the invention concerns the use of thenovel peptide as an inhibitor of ecdysone biosynthesis. Ecdysone is ahormone found in flies and mosquitoes which regulates molting, growth,and gametogenesis. Thus, Neb-TMOF falls into a new class of biorationalinsecticides that are, advantageously, natural, target-specific, and, incontrast to the early organic insecticides, e.g., DDT, readily degradedin the environment and do not cause pollution problems.

Also included in this invention are addition salts, complexes, orprodrugs such as esters of the peptides of this invention, especiallythe nontoxic pharmaceutically or agriculturally acceptable acid additionsalts. The acid addition salts are prepared in standard manner in asuitable solvent from the parent compound and an excess of an acid, suchas hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic,succinic, ethanedisulfonic or methanesulfonic acids. The formulas haveweak acidic groups (carboxyl groups); thus, esterification at thesegroups to form derivatives such as the methyl or ethyl esters, can beprepared by standard procedures. The term "prodrug" is understood in theart to mean a compound chemically related to the subject compound thatis converted into the subject compound by metabolic process within thebody (biotransformation).

In a further embodiment, the N-terminus and C-terminus of the peptidescan be blocked to further inhibit proteolysis by metabolic enzymes.Derivation of peptides to block the N-terminus or C-terminus is known inthe art. For example, the N-terminus can be acetylated by methods knownto those of ordinary skill in the art; the C-terminus can be amidated asis well known in the art.

The novel peptides can also be synthesized wherein at least one of theamino acids is in the D-conformation, as opposed to the naturallyoccurring L-rotation conformation. The presence of D-conformation aminoacids can inhibit the ability of proteases to degrade the peptides ofthe Subject invention.

Also, derivation of these compounds with long chain hydrocarbons willfacilitate passage through the cuticle into the pest body cavity.Therefore, a further embodiment of the subject invention pertains tocompositions comprising the peptides bound to lipids or other carriers.

A further aspect of the subject invention pertains to nucleic acid,e.g., DNA, sequences encoding the peptides disclosed herein. These DNAsequences can easily be synthesized by a person skilled in the art. Thesequences may be used to transform an appropriate host to confer uponthat host the ability to express the novel peptides. Hosts of particularinterest include bacteria, yeasts, insect viruses, and plants. For eachof these hosts, the DNA sequences may be specifically designed by aperson skilled in the art to utilize codons known to be optimallyexpressed in the particular hosts. Advantageous promoters can alsoeasily be utilized. Bacteria, yeasts, and viruses each may be used toproduce peptide for further use, or these hosts can be used as vehiclesfor direct application of the peptide to the target pest. Plants can betransformed so as to make the plant effective in controlling or killinga target pest species which feeds on that plant. Methods fortransforming plant cells utilizing, for example agrobacteria or viruses,are well known to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Stereoview of Neb-TMOF represented by a stick model on aMacintosh II work station connected to a VAX using SYBYL molecularmodeling software version 5.2 (Tripos Associates Inc., St. Louis, Mo.).The N-terminus is labeled Asn1 (top) and the C-terminus is His6(bottom).

FIG. 2. Comparison between the amount of trypsin in the gut (μg/gut) andfollicle development in the ovary at different times after liver feeding(0 hr). Egg stages are according to Pappas and Fraenkel (1978).

FIG. 3. First C₁₈ column (Deltapak) chromatography of 1,000 ovaries. Thehatched area represents inhibition of oocyte development and trypsinbiosynthesis.

FIG. 4. Last HPLC-purification step (v) using C₁₈ column. Neb-TMOF waseluted at 25.6% acetonitrile. The diode array scan indicates absorbanceonly at 214 nm.

FIG. 5. Inhibition of trypsin biosynthesis in the gut by different dosesof synthetic Neb-TMOF (- - -). Significant inhibition (- - -) (P<0.05)was obtained in vivo at concentrations of 10⁻⁸ M and 10⁻⁹ M.

FIG. 6. Comparison between the effect of Neb-TMOF (10⁻⁹ M) on flies by:injection (inj), adding TMOF to the incubation medium (inc), orinjecting flies with water (controls C).

FIG. 7. Effect of Neb-TMOF on vitellogenin biosynthesis in flies.Females were injected with Neb-TMOF (10⁻⁹ M) and hemolymph (0.5 μl) wasassayed for vitellogenin subunits using SDS-PAGE (5-15% gradient). Lane(1) Male's hemolymph; Lane (2) Hemolymph of control females; Lane (3)Hemolymph of Neb-TMOF injected females; Lane (4) Standards:phosphorylase b (94 kDa), Bovine Serum Albumin (67 kDa), Ovalbumin (43kDa), Carbonic Anhydrase (30 kDa), Soybean trypsin inhibitor (20.1 kDa)and α-lactalbumin (14.4 kDa).

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns novel peptides that inhibit digestion intarget pests. Specifically exemplified is the use of the peptides withblood-ingesting insects such as the flesh fly (genus Neobellieria). Oneembodiment of the subject invention is a hexapeptide, designatedNeb-TMOF, which has the amino acid sequence shown in SEQ ID NO. 1.Having only six amino acids, it is only about 50% the size of thepreviously-described TMOF hormone found in mosquitoes. Advantageously,with this substantial truncation as compared to the mosquito factor, thepeptide retains biological activity and has important practicaladvantages because it is rapidly absorbed and less susceptible toproteolysis. Also encompassed within the scope of this invention arecertain modifications of these peptides.

The one-letter symbol for the amino acids used herein is well known inthe art. For convenience, the relationship of the three-letterabbreviation and the one-letter symbol for amino acids is as follows:

    ______________________________________                                        Ala       A             Leu    L                                              Arg       R             Lys    K                                              Asn       N             Met    M                                              Asp       D             Phe    F                                              Cys       C             Pro    P                                              Gln       Q             Ser    S                                              Glu       E             Thr    T                                              Gly       G             Trp    W                                              His       H             Tyr    Y                                              Ile       I             Val    V                                              ______________________________________                                    

The novel peptides of the invention can be prepared by well-knownsynthetic procedures. For example, the peptides can be prepared by thewell-known Merrifield (1963) solid support method. This procedure,though using many of the same chemical reactions and blocking groups ofclassical peptide synthesis, provides a growing peptide chain anchoredby its carboxyl terminus to a solid support, usually cross-linkedpolystyrene or styrenedivinylbenzene copolymer. This method convenientlysimplifies the number of procedural manipulations since removal of theexcess reagents at each step is effected simply by washing of thepolymer.

Alternatively, these peptides can be prepared by use of well-knownmolecular biology procedures. Nucleic acid, e.g., DNA, sequencesencoding the peptides of the invention can be synthesized readilybecause the amino acid sequences are disclosed herein. These nucleicacid sequences are a further aspect of the subject invention. Thesegenes can be used to genetically engineer, for example, bacteria, insector plant viruses, plant cells, or fungi for synthesis of the peptides ofthe invention.

The insect cell line Sf9 (Spodoptera frugiperda), deposit number ATCCCRL 1711, is available from the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 USA. Baculovirus Autographacalifornica nuclear polyhedrosis virus (AcNPV) is available from TexasA&M University, Texas Agricultural Experiment Station, College Station,Tex. 77843, and has been described in Smith and Summers (1978; 1979).

Other nuclear polyhedrosis viruses (See World Health OrganizationTechnical Report No. 531) such as Spodoptera frugiperda (Sf MNPV),Choristoneura furniferana (Cf MNPV) (Smith and Summers, 1981), orSpodoptera littoralis (S1 NPV) (Harrap et al., 1977) can be used insteadof Autographa californica NPV. Other insect cell lines can also besubstituted for Spodoptera frugiperda (Sf9), for example, Trichoplusiani (Volkman and Summers, 1975), Spodoptera exigua, Choristoneurafurniferana (Smith and Summers, 1981) and Spodoptera littoralis (Harrapet al., 1977).

Viruses can also be used as expression vectors in transfected plantcells, which then produce a desired protein. For example, a heterologousnucleic acid of tobacco mosaic virus (TMV) can be used to express thesubject peptide in a plant cell. See Donson et al., 1991. The subjectpeptide, which is expressed by the virus-transfected plant cell, can beingested by the target insect pest when the pest feeds on the plant. Theingested peptide can then inhibit production of oocytes in the insectpest as described herein.

The various methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art and aredescribed, for example, in U.S. Pat. Nos. 5,011,909 and 5,130,253. Thesepatents are incorporated herein by reference. These procedures are alsodescribed in Maniatis et al., 1982. Thus, it is within the skill ofthose in the genetic engineering art to extract DNA from microbialcells, perform restrictions enzyme digestions, electrophorese DNAfragments, tail and anneal plasmid and insert DNA, ligate DNA, transformcells, e.g., E. coli or plant cells, prepare plasmid DNA, electrophoreseproteins, and sequence DNA.

Treatment by injection of the compounds of the invention into adultfemale flesh flies after a blood meal stops egg development, thusrendering the female flesh fly sterile and unable to reproduce. Also,using known techniques of molecular biology, fly larvae can be fedgenetically engineered bacteria producing oostatic hormone and infectother insect larvae with bacteria or viruses containing the oostaticgene, making them unable to digest their food and subsequently starve todeath. A variety of insect viruses, including baculoviruses andentomopoxviruses, are known to those skilled in the art. The productionof the claimed peptide compounds by bacteria or virus would beresponsible for the starvation activity in larvae and sterilization inadults. This type of treatment of blood-ingesting insect larvae isanalogous to the use of bacteria to control insect populations.

In applications to the environment of the target pest, the transformantstrain can be applied to the natural habitat of the pest. Thetransformant strain will grow in the pest upon ingestion, whileproducing the peptide(s) which will have a deleterious effect onproteolytic enzymes biosynthesis and the ova. The organism may beapplied by spraying, soaking, injection into the soil, seed coating,seedling coating or spraying, or the like. Where administered in theenvironment, concentrations of the organism will generally be from 10⁶to 10¹⁰ cells/ml, and the volume applied per hectare will be generallyfrom about 0.1 oz. to 2 lbs or more. Where administered to a plant paninhabited by the target insect, the concentration of the organism willusually be from 10³ to 10⁶ cells/cm².

In aquatic environments, insect control may be attained below thesurface by varying the lipid content of the transformant microorganismstrain. It is known that indigenous aquatic algae float due to theirlipid content. A variation in lipid content will allow the transformantstrain to be distributed at desired depths below the water surface.

For commercial formulations, the organisms may be maintained in anutrient medium which maintains selectivity and results in a low rate ofproliferation. Various media may be used, such as yeast extract orL-broth. Once the organism is to be used in the field, thenon-proliferating concentrate may be introduced into an appropriateselective nutrient medium, grown to high concentration, generally fromabout 10⁵ to 10⁹ cells/ml and may then be employed for introduction intothe environment of the blood-ingesting insect.

Also, the genetic material of the subject invention, includingnucleotide sequences of Neb-TMOF and its various analogs, can be used totransform plants, thereby conferring plant resistance to those plants.Materials and methods for transforming plant cells are well known tothose skilled in the art.

The subject Neb-TMOF was purified from Neobellieria using highperformance liquid chromatography (HPLC). Preferably, the HPLC procedurefor purifying the subject peptide is conducted using five chromatographycolumns, following preparation of the sample. The in vivo bioassay andthe dose-response curve using synthetic Neb-TMOF suggests that it has animportant physiological role in directly controlling digestion andindirectly, the female reproductive cycle. Experiments conducted usingNeb-TMOF indicated that it is sixfold more active in Neobellieria thanthe mosquito TMOF. Computer modelling of the subject peptide using themolecular modelling program SYBYL (Tripos Associates, St. Louis Mo. USA)(Borovsky et al. 1993a) showed that the alpha-helix, which is formed bythe six consecutive prolines in mosquito TMOF is absent in Neb-TMOF(FIG. 1).

The Neb-TMOF inhibits the de novo biosynthesis of trypsin. Similarly,allatostatins can block juvenile hormone biosynthesis in the corporaallata of the cockroach Diploptera, by inhibiting the biosynthesis ofkey enzymes in the juvenile hormone biosynthetic pathway. In addition,Neb-TMOF can affect egg development in Neobellieria and Calliphora byinhibiting the biosynthesis of ecdysone.

Materials and Methods

Insect breeding and egg development. Fries were reared as described byHuybrechts and De Loof (1977). During the first three days after adulteclosion flies were fed sugar and water. From the fourth day, flies werefed on slices of beef liver. To maintain a synchronous ovariandevelopment, 2-day-old females were separated and individually caged inplastic tubes, containing wet cotton. At day 4, females wereanesthetized with CO₂, immediately injected with test peptides in 2 μlof saline and fed liver coated with sugar crystals. The liver-sugarcombination attracted the flies resulting in a faster food intake andovarian development. Oocyte length was measured with an ocularmicrometer under a dissecting microscope.

Effect of Neb-TMOF on trypsin biosynthesis. Six hours after injectingNeb-TMOF or analogs, guts were removed and analyzed for trypsinbiosynthesis (Borovsky and Schlein, 1988), or kept frozen at -20° C.until use. Trypsin was measured in the presence of [³H]-diisopropylfluorophosphate (DFP) (New England Nuclear, specificactivity 10 Ci/mmol). In the presence of serine proteases [³ H]DFP isconvened into [³ H]-diisopropyl phosphoryl- ([³ H]DIP) trypsinderivatives which are measured by liquid scintillation. The incubationmedium contains 5 mM tosylamide-2-phenylethyl ketone (TPCK) achymotrypsin inhibitor, thus making the test specific for trypsin.Because the amino acid sequence of Neobellieria trypsin is not known,the term trypsin-like enzyme is more appropriate. However, for reasonsof simplicity, the term trypsin is used. Individual guts werehomogenized, centrifuged and the supernatant removed and an aliquot (0.1gut equivalent) was incubated with [³ H]DFP for 18 hours at 5° C.Standard curves were obtained with trypsin type III from bovine pancreas(Sigma Chemical Co., St. Louis, Mo.) (Borovsky and Schlein 1988). Ineach experiment 2 control groups were used: (a) uninjected and, (b)injected with 10⁻⁷ M methionine-enkephalin (H-YGGFM-OH), a pentapeptidewithout an effect on trypsin-like activity. No significant differenceswere found between the 2 control groups. Statistical significance wascalculated using Microstat software. Chymotrypsin-like concentrationswere monitored by the same method in the presence of 5 mMtosyl-L-lysine-chloromethyl ketone (TLCK) instead of TPCK to inhibittrypsin-like enzymes (Borovsky and Schlein 1988).

Electrophoresis and fluorography. [³ H]DIP-trypsin-like derivatives of0.04 gut equivalent and 20 μl sample buffer were run by electrophoresison 12.5% polyacrylamide gel for 1 hour at 20 mA and for 3 hours at 30 mA(Biorad protean 32 CM) (Laemmli, 1970; Borovsky and Schlein, 1988). Gelswere stained for 20 minutes with Coomassie Brilliant Blue R250 anddestained overnight with methanol/H₂ O/acetic acid (40/50/10), andwashed 2 times for 30 minutes in 100 ml dimethylsulfoxide (DMSO). Gelswere then incubated for 2 h in 200 ml DMSO containing 44 g2,5-diphenyloxazole (PPO) (Janssen Chimica, Belgium), rinsed withdistilled water for 1 hour, dried for 2 hours in a Biorad slab gel dryerat 80° C. and exposed to a Hyperfilm-MP (Amersham) for 2 days at -70° C.Vitellogenin was separated on a 5-15% SDS gradient gel (Huybrechts andDe Loof 1977).

Tissue extraction and High Performance Liquid Chromatography. Latevitellogenic ovaries stage 4C (10,000 pairs) were dissected andimmediately placed in methanol/water/acetic acid (90:9:1) solution onice. The ovaries were homogenized, centrifuged for 30 minutes at 9,820 gand 4° C. and sonicated for 2 minutes (MSE Soniprep 150 Sonicator).Methanol was evaporated and the aqueous extract was re-extracted withethyl acetate and n-hexane. Organic solvents were decanted and theaqueous solution was dried in siliconized round bottom flasks. Extractswere then prepurified on Megabond Elute C₁₈ cartridges (Varian).Cartridges were activated with acetonitrile/H₂ O/trifluoroacetic acid(TFA) (80/19.9/0.1) and afterwards rinsed with aqueous 0.1% TFA. Sampleswere redissolved in start solution and subsequently eluted with 50% and80% acetonitrile containing 0.1% TFA. Columns and operation conditionsfor High Performance Liquid Chromatography (HPLC) on BeckmanProgrammable Solvent Module 126 connected with a Diode Array DetectorModule 168 (Gold system) were:

(i) Deltapak RCM column (25×100 mm) (Waters), solvent A, 0.1% TFA inwater; solvent B, 80% acetonitrile in 0.1% aqueous TFA. Column elutionconditions: 100% A for 8 minutes, linear gradient to 75% B in 60minutes, flow rate 8 ml/min.

(ii) Supelco C₈ column (4.6×150 mm), cfr. (i) but flow 1.5 ml/min.

(iii) a refilled stainless steel column (4.6×250 mm) with a new phenylcontent (Nucleosil 7 C6H5, Machery-Nagel), cfr. (ii).

(iv) cfr. (iii) but A, 0.1% heptafluorobutyric acid (HFBA) and B 80%acetonitrile in 0.1% aqueous HFBA.

(v) cfr. (iii) and (iv), except elution was carried out with 80%acetonitrile in 0.1% aqueous HFBA.

Absorbance was followed at 214 nm. During the first two columnprocedures, fractions were collected every minute. Afterwards, peakswere collected manually. At each purification step aliquots from eachfraction were removed, dried under N₂ and rehydrated in HPLC water.Adjacent active fractions were pooled, dried under N₂ and redissolved inthe buffer used for the next purification step.

Mass and sequence determination. Accurate monoisotopic molecular massesof peptides were obtained by liquid secondary ionization massspectrometry (LSIMS) on a quadrupole Fourier transform mass spectrometer(QFTMS) constructed in our laboratory and previously described (Hunt etal. 1987). Samples to be analyzed were dissolved in acidic solution, andan aliquot containing 1-10 pmol of peptide was added to a gold-plated,stainless steel, probe tip (2 mm diameter) in addition to 1 μl of matrix(3:1, monothio-glycerol:glycerol). The probe tip was then evacuated andinserted into ion source region of the QFTMS. Peptides were sputteredout of the liquid matrix into the gas phase by bombardment of the samplewith a pulsed beam of 10 keV Cs⁺ generated by a cesium ion gun (Antek,Palo Alto, Calif.). This method of ionization (LSIMS) produces apopulation of singly charged, peptide ions, (M+H)⁺, characteristic ofthe peptides in the sample.

Collision activated dissociation (CAD) mass spectra for sequenceanalysis were recorded on a Finnigan TSQ-70, triple quadrupole massspectrometer equipped with a Finnigan electrospray ionization source(Finnigan-MAT, San Jose, Calif.). Operation of this instrument has beenpreviously described (Hunt et al. 1986, 1991). Sample aliquots dissolvedin acidic solutions were injected into the electrospray ionizationsource from a fused silica, microcapillary HPLC column with an insidediameter of 75 microns and a length of 70 cm. The last 10 cm of thecolumm was filled with a C-18 packing material. Peptides were eluted ata flow rate of 0.5-1 μl/min with a 10 minute, linear gradient of 0-80%acetonitrile in 0.1M acetic acid. Sequence analysis is conductedroutinely at the low to subpicomole level (3-0.3 pmol).

Esterification of the carboxylic acid moieties of peptides wasaccomplished by the addition of 20-50 μl of 2N methanolic HCl tolyophilized HPLC fractions containing the peptides of interest (Hunt etal. 1986). Esterification proceeded for 90 minutes, and the reagent wasremoved by vacuum centrifugation of the sample to dryness. Modifiedpeptides were resuspended in an appropriate volume of 5% acetic acid.

N-acetylation of the N-terminus and lysine residues can be performeddirectly on the microcapillary HPLC column. Peptides were loaded ontothe microcapillary column and washed with water for 3 minutes. Reagent,1 μl acetic anhydride is 100 μl of 200 mM ammonium acetate, pH 8.0, wasthen loaded onto the column for 3 minutes (one column volume). Thecolumn was washed with water again for 3 minutes before starting theHPLC program.

Automated Edman sequencing was performed using an Applied Biosystems(Foster City, Calif.) model 473A pulsed liquid protein sequencer and120A analyzer operated according to standard procedures.

Peptide synthesis. Peptides were synthesized using solid phase9-fluoroenylmethoxycarbonyl (Fmoc) methodology. The coupling of theC-terminal amino acid to the resin was catalyzed by4-dimethylaminopyridine. Chain elongation and coupling of amino acidswere done using H-hydroxybenzotriazole-monohydrate. Twenty percentpiperidine in dimethylformamide was used to remove the protecting Fmocgroup of each amino acid. TFA was used to cleave the peptide and removeprotecting group from side chains. The peptide was then dried by rotaryevaporation and prepurified on a Sep-Pak column (Waters Associates)using the same conditions for the Megabond Elut column. The syntheticpeptide was then repurified on a Waters Superpac Pep S C₂ /C₁₈ 5 μmcolumn.

Amino acid analysis. The purified synthetic peptide was hydrolyzed with6N HCl at 120° C. for 18 hours. The amino acid content of the peptidewas determined using ion exchange separation on a Kontron Chromakon 400amino acid analyzer followed by postcolumn derivatization withninhydrin. Absorbance was followed at 570 nm.

Methyl ester formation. A standard solution of 2N HCl in methanol wasprepared by adding 800 μl of acetyl chloride dropwise, with stirring, to5 ml of methanol. After 5 minutes incubation at room temperature, a 100μl aliquot of the reagent was added to the lyophilized peptide sample.The sample was esterified for 2 hours at room temperature and thesolvent removed by lyophilization.

Peptide N-acetylation. The peptide was dissolved in 50 μl of 50 mMammonium bicarbonate (pH 8.0) and 50 μl of freshly prepared acetylationreagent was added to this solution. Acetylation reagent was prepared byadding 100 μl of acetic anhydride to 300 μl of dry methanol andlyophilizing the mixture after allowing it to stand 15 minutes.Acetylated peptide was analyzed directly without further purification.

Manual Edman degradation. Manual Edman degradations were performed aspreviously described (Tarr, 1977) and modified for use with massspectrometry (Hunt et al., 1986).

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1--Synthesis of Trypsin in the Gut

After a protein meal a steep rise in trypsin biosynthesis was observedreaching a peak at 6 hours and then declined to a minimum at 72 hours(FIG. 2). Egg development followed trypsin biosynthesis in the midgut(FIG. 2). In the males, cycles of increase and decrease in trypsinbiosynthesis after a liver meal are less pronounced than in the female.At 96 hours after the eggs descended into the uterus, trypsinbiosynthesis also increased. Thus, trypsin synthesis is cyclical andclosely correlated with oocyte-development. Since chymotrypsin-likeenzymes activity is less than 5% of trypsin-like activity no attempt wasmade to follow their biosynthesis.

EXAMPLE 2--Role of the Ovary in Modulating Trypsin Biosynthesis

Injection of a crude extract of ovaries (2 to 5 equivalents; 4C stage)after acid treatment into 4-day-old females immediately before a livermeal, inhibited 50% of trypsin biosynthesis 6 hours later when comparedwith controls. In ovariectomized flies the level of trypsin-like enzymesremains at a higher level, whereas, in sham operated or untreatedcontrols, trypsin biosynthesis drops rapidly (FIG. 2). The initialincrease in trypsin biosynthesis in ovariectomized females is lesspronounced than in controls, and could be due to the operation. Theseresults indicate that the ovary modulates trypsin biosynthesis in thefly.

EXAMPLE 3--HPLC Purification of Neb-TMOF

Ten Megabond Elut columns were used to prepurify the ovarian extract.The fraction eluted with 50% acetonitrile-water, 0.1% TFA significantlyinhibited oocyte growth at 2 ovary equivalents. After the first HPLCcolumn, Neb-TMOF activity was eluted at 20-21% acetonitrile (fractions25-27) (FIG. 3). Injection of 2 ovary equivalents caused significantinhibition (P<0.003) of oocyte growth at 24 hours and significantdecrease in the amount of trypsin at 6 hours after liver feeding. Toconserve Neb-TMOF, fractions were monitored from this step onwards onlyfor the effect on trypsin biosynthesis. Neb-TMOF was then purified on C₈and 2 phenyl columns eluted with acetonitrile 0.1% TFA. A third phenylcolumn was then eluted with acetonitrile 0.1% HFBA. During the 4 columnpurification steps, Neb-TMOF activity was found in fractions eluted at8, 10, 18 and 26% acetonitrile, respectively. Inhibition of trypsinbiosynthesis during the 5 column purification steps was 44, 38, 43, 44and 20% (P<0.05) using 1.25, 3.7, 10, 10 and 38.5 ovary equivalents,respectively. After the fifth column (FIG. 4), the peptide showedapparent homogeneity and was sequenced. Because of losses of biologicalactivity due to sample loss during the five step purification procedure,higher number of ovary equivalents were needed at the last step toinhibit trypsin biosynthesis.

EXAMPLE 4--Sequence Determination of the Purified Neb-TMOF

Automated Edman sequence analysis of the isolated Neb-TMOF yielded anambiguous analysis even though it had been subjected to five stages ofpurification. Aliquots of the sample were then analyzed by massspectrometric analyses. Molecular masses were measured at 695.3, 709.4,and 737.4 for the free acid, methyl ester, and acetylated peptiderespectively. Portions of purified Neb-TMOF and the above derivativeswere then subjected to microcapillary HPLC interfaced directly to theelectrospray ionization source of a Finnigan triple quadrupole massspectrometer. Collision activated dissociation mass spectra wereobtained for the (M+H)⁺ ions of the free acid, methyl ester andacetylated peptides. Interpretation of this data led to the proposedsequence, NH₂ -Asn-Pro-Thr-Asn-Leu(Ile)-His-COOH. Leu and Ile haveidentical mass and cannot be distinguished under the experimentalconditions employed. Data obtained form the automated Edman sequenceanalysis was used to assign the fifth residue as leucine. The peptideNH₂ -Asn-Pro-Thr-Asn-Leu-His-COOH was then synthesized, purified onreversed phase C₁₈ column and subjected to amino acid analysis whichconfirmed its primary structure. The synthetic hormone coeluted with thenatural Neb-TMOF and was used in the biological studies.

EXAMPLE 5 --Activity of the Synthetic Peptide, Dose Response and Mode ofAction

To calculate the concentration of Neb-TMOF in the hemolymph afterinjection, we estimated that Neobellieria has 20 μl of hemolymph, whichcorresponds to 25% of the total weight of the fly. Injection of Neb-TMOFat concentrations of 10⁻⁸ and 10⁻⁹ M into 4-day-old females resulted ina 35% and 60% inhibition of trypsin biosynthesis (FIG. 5). No inhibitionwas detected at concentrations of 10⁻¹¹ M. To find out if Neb-TMOFinterferes with the conversion of [³ H]DFP to [³ H]DIP-trypsinderivatives, guts were removed from flies 6 hours after a liver meal,homogenized, centrifuged and the supernatant incubated for 18 hours at4° C. in the presence and absence of Neb-TMOF. No reduction in [³H]DIP-trypsin derivatives was observed when compared with the controls(FIG. 6). To find out whether Neb-TMOF inhibits the de novo biosynthesisof trypsin, four-day-old females were injected with Neb-TMOF (finalconcentration 10⁻⁹ M) and then fed a liver meal. Guts were removed 6 hlater, homogenized, centrifuged, and supernatants incubated with [³H]DFP for 18 h at 4° C. and analyzed for [³ H]DIP-trypsin-likederivatives. A 51% reduction in trypsin-like enzymes was observed afterinjection of 10⁻⁹ M Neb-TMOF as compared with controls (FIG. 6). Theseexperiments demonstrate that Neb-TMOF inhibits trypsin biosynthesis andnot trypsin activity as soybean trypsin inhibitor, N-tosyl-L-lysinechloromethyl ketone (TLCK), which bind to the active site of trypsin andinhibit enzyme activity.

EXAMPLE 6--Inhibition of Vitellogenin Synthesis by Neb-TMOF

Four-day-old females were injected with synthetic Neb-TMOF at finalconcentration of 10⁻⁹ M. Twenty-four hours later, hemolymph wascollected and subjected to PAGE. As shown in FIG. 2, low level ofvitellogenin was found in injected females as compared with non-injectedcontrols. The three yolk polypeptides of vitellogenin were affected(FIG. 7). The decrease in vitellogenin in the hemolymph is probably dueto the inhibition of trypsin biosynthesis and decrease in proteindigestion in the gut. The Neb-TMOF can also affect the fat body, whichis the major site of yolk polypeptide biosynthesis in Neobellieria.

EXAMPLE 7--Effect of Neb-TMOF on Ecdysteroid Synthesis

In addition to the direct effect of Neb-TMOF (SEQ ID NO. 1) ontrypsin-like biosynthesis in both adults and larvae and the indirecteffect of the peptide on oocyte growth, we observed activity of thesubject peptide on ecdysteroid, e.g., ecdysone, biosynthesis and/orrelease. In insects, ecdysteroids are well known steroid hormonesresponsible for controlling processes involved in activation of genesfor cuticle formation, stimulation of vitellogenin synthesis by the fatbody, spermatocyte growth, and induction of diapause. The role ofecdysteroids in reproduction, especially in Diptera, is well known, andecdysteroids were proved to initiate the synthesis of the femalespecific vitellogenin in males by injection of Neobellieria bullata(Huybrechts and De Loof, 1977).

The peptide of the subject invention was demonstrated to inhibit invitro ecdysone biosynthesis by the ring glands of larvae of the fleshflies Calliphora and Neobellieria in a dose-dependent manner. The EC₅₀was shown to be 5×10⁻⁹ M. More than 98% of ecdysone biosynthesis wasinhibited at 10⁻⁷ M. This activity is called prothoracico-inhibitingactivity (PTIH). The PTIH effect by Neb-TMOF was shown to be immediateand reversible. A ten picomole quantity of the peptide per larva reducesin vivo the ecdysteroid titer by up to a factor of four within 18 hours.

Further recent studies showed the existence of the subject peptide inlarvae and pupae of the flesh files. Thus, the subject peptide can haveimportant physiological roles in ecdysteroid regulation and, hence, aneffect on molting, growth, and gametogenesis. Because of the role ofecdysteroids on vitellogenin synthesis by the fat body, the inhibitingeffect of Neb-TMOF on oocyte growth can be multiple. First, the supplyof amino acids for vitellogenin production is prohibited by Neb-TMOF'seffect on inhibiting digestion. Second, Neb-TMOF can arrest synthesis ofecdysone, the trigger for vitellogenin synthesis.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

References

Patents:

Borovsky, D., D. A. Carlson, U.S. Pat. No. 5,011,909, issued Apr. 30,1991.

Borovsky, D., D. A. Carlson, U.S. Pat. No. 5,130,253, issued Jul. 14,1992.

Publications

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Adams, T. S. (1976) "The ovaries, ring gland and neurosecretion duringthe second gonotrophic cycle in the housefly, Musca domestica," Gen.Comp. Endocrinol. 30:69-76.

Borovsky, D., B. R. Thomas, D. A. Carlson, L. R. Whisenton, M. S. Fuchs(1985) "Juvenile hormone and 20-hydroxyecdysone as primary and secondarystimuli of vitellogenesis in Aedes aegypti," Arch. Insect Biochem.Physiol. 2:75-90.

Borovsky, D., Y. Schlein (1988) "Quantitative determination oftrypsinlike and chymotrypsinlike enzymes in insects," Arch. InsectBiochem. Physiol. 8:249-260.

Borovsky, D., D. A. Carlson, P. R. Griffin, P. R., J. Shabanowitz, D. F.Hunt (1990) "Mosquito oostatic factor: a novel decapeptide modulatingtrypsin-like enzyme biosynthesis in the midgut," FASEB J. 4:3015-3020.

Borovsky, D., D. A. Carlson, I. Ujvary (1992a) "In vivo and in vitrobiosynthesis and metabolism of methyl farnesoate, juvenile hormone IIIand juvenile hormone III acid in the mosquito Aedes aegypti," J. Med.Ent. 29:619-629.

Borovsky, D., C. A. Powell, D. A. Carlson (1992b) "Development ofspecific RIA and ELISA to study trypsin modulating oostatic factor inmosquitoes," Arch. Insect Biochem. Physiol. 21:13-21.

Borovsky, D., D. A. Carlson, P. R. Griffin, J. Shabanowitz, D. F. Hunt(1993a) "Mass spectrometry and characterization of Aedes aegypti trypsinmodulating oostatic factor (TMOF) and its analogs," Insect Biochem. Mol.Biol. 27:703-712.

Borovsky, D., D. A. Carlson, P. R. Griffin, J. Shabanowitz, D. F. Hunt(1993c) "Sequencing and characterization of Aedes aegypti trypsinmodulating oostatic factor," In Borovsky, D., A. Spielman (eds) HostRegulated Developmental mechanisms in Vector Arthropods, Proceedings ofthe Third Symposium, University of Florida, Vero Beach, Fla., USA, pp.36-47.

Borovsky, D., Q. Song, M. Ma, D.A. Carlson (1993d) "Biosynthesis,secretion, and cytoimmunochemistry of trypsin modulating oostatic factorof Aedes aegypti," Arch. Insect Biochem. Physiol. (In press).

Domson, J., C. M. Kearney, M. E. Hilf, W. D. Davison (1991) "Systemicexpression of a bacterial gene by a tobacco mosaic virus-based vector,"PNAS (Genetics) 88:7204-7208.

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Goltzene, F., M. Lageaux, M. Charlet, J. A. Hoffman (1978) "The folliclecell epithelium of maturing ovaries of Locusta migratoria: a newbiosynthetic tissue for ecdysone," Hoppe-Seyler's Z. Physiol. Chem.359:1427-1434.

Hagedorn, H. H., J. D. O'Connor, M. S. Fuchs, B. Sage, D. A Schlaeger,M. K Bohm (1975) "The ovary as a source of alpha-ecdysone in an adultmosquito," Proc. Natl. Acad. Sci USA 72:3255-3259.

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Woodhead, A. P., B. Stay, S. L. Seidel, M. A. Khan, S. S. Tobe (1989)"Primary structure of four allatostatins: Neuropeptide inhibitors ofjuvenile hormone synthesis," Proc. Natl. Acad. Sci USA 86:5997-6001.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AsnProThrAsnLeuHis                                                            15                                                                        

We claim:
 1. A substantially pure peptide having the amino acid sequenceshown in SEQ ID NO.
 1. 2. The peptide, according to claim 1, wherein theN-terminus of said peptide is acetylated.
 3. The peptide, according toclaim 1, wherein the C-terminus of said peptide is amidated.
 4. Thepeptide, according to claim 1, comprising a D-amino acid.
 5. Thepeptide, according to claim 1, wherein said peptide is bound to a lipid.6. A pesticide composition comprising a peptide and a carrier, whereinsaid peptide has the amino acid sequence shown in SEQ ID NO.
 1. 7. Thepesticide, according to claim 6, wherein the C-terminus of said peptideis amidated.
 8. The pesticide, according to claim 6, wherein theN-terminus of said peptide is acetylated.
 9. The pesticide, according toclaim 6, wherein said peptide is bound to a lipid.